IP Library › Patent Application 18629661

Patent Application

App. No. 18/629,661

MULTIPLEXED TARGETED AMPLIFICATION FOR MUTATION DETECTION

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Patent No.: US None
App. No.: 18/629,661

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Abstract

Disclosed herein, inter alia, are compositions and methods for amplification and sequencing of nucleic acid molecules including, for example, genetic mutations.

Claims (80)

1 . A solid support comprising:

a first oligonucleotide attached to said solid support comprising a first sequence capable of hybridizing to a first endogenous sequence of a target polynucleotide;

a second oligonucleotide attached to said solid support comprising a second sequence capable of hybridizing to the complement of a second endogenous sequence of said target polynucleotide; and

a target polynucleotide hybridized to said first oligonucleotide, wherein said target polynucleotide comprises the first endogenous sequence, a target sequence, and the second endogenous sequence.

2 . The solid support of claim 1 , wherein the target polynucleotide comprises an NRAS gene, or a portion thereof, a MLF2 gene, or a portion thereof, a KRAS gene, or a portion thereof, a TP53 gene, or a portion thereof, a ERBB2 gene, or a portion thereof, a IDH1 gene, or a portion thereof, a PIK3CA gene, or a portion thereof, a KIT gene, or a portion thereof, a PIK3R1 gene, or a portion thereof, a NPM1 gene, or a portion thereof, a EGFR gene, or a portion thereof, a BRAF gene, or a portion thereof, or a JAK2 gene, or a portion thereof;

a mutated NRAS gene, or a portion thereof, a mutated MLF2 gene, or a portion thereof, a mutated KRAS gene, or a portion thereof, a mutated TP53 gene, or a portion thereof, a mutated ERBB2 gene, or a portion thereof, a mutated IDH1 gene, or a portion thereof, a mutated PIK3CA gene, or a portion thereof, a mutated KIT gene, or a portion thereof, a mutated PIK3R1 gene, or a portion thereof, a mutated NPM1 gene, or a portion thereof, a mutated EGFR gene, or a portion thereof, a mutated BRAF gene, or a portion thereof, or a mutated JAK2 gene, or a portion thereof.

3 . The solid support of claim 1 , further comprising a plurality of first oligonucleotides attached to said solid support comprising said first sequence; and a plurality of second oligonucleotides attached to said solid support comprising said second sequence.

4 . The solid support of claim 1 , wherein said target sequence is about 60 nucleotides to about 150 nucleotides.

5 . The solid support of claim 1 , wherein said first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 7; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:9; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:11; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:13; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 15; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:17; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:19; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:21; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:23; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:25; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 27; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:29; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:31; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:33; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:35; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:37; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:39; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:41; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:43; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:45; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:47; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 49; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:51; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:53; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:55; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:57; or at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:59.

6 . The solid support of claim 1 , wherein said first sequence comprises SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO: 41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO: 53, SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:59.

7 . The solid support of claim 1 , wherein said second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 8; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:10; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 12; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 14; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:16; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 18; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:20; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:22; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:24; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:26; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 28; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:30; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:32; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:34; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:36; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:38; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:40; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:42; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:44; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:46; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:48; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 50; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:52; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:54; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:56; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:58; or at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:60.

8 . The solid support of claim 1 , wherein said second sequence comprises SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO: 52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, and SEQ ID NO:60.

9 . The solid support of claim 1 , wherein the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 7; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:8;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 9; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 10;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 11; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 12;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 13; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 14;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 15; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 16;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 17; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 18;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 19; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 20;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 21; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 22;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 23; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 24;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 25; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 26;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 27; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 28;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 29; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 30;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 31; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 32;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 33; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 34;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 35; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 36;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 37; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 38;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 39; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 40;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 41; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 42;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 43; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 44;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 45; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 46;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 47; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 48;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 49; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 50;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 51; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 52;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 53; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 54;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 55; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 56;

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 57; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 58; or

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 59; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 60.

10 . The solid support of claim 1 , wherein said solid support comprises a plurality of particles, wherein each of said plurality of particles comprises said first oligonucleotide and said second oligonucleotide.

11 . The solid support of claim 1 , wherein said solid support comprises a plurality of wells.

12 . The solid support of claim 11 , wherein each well is about 5 mm to about 8 mm in diameter.

13 . The solid support of claim 11 , wherein each well comprises a plurality of nanowells, wherein each nanowell is about 0.1 μm to about 2 μm in depth, and wherein each nanowell is about 0.1 μm to about 2.0 μm in diameter.

14 . The solid support of claim 11 , wherein the solid support comprises about 0.2 wells to about 4.0 wells per μm 2 .

15 . A microfluidic device comprising the solid support of claim 1 .

16 . A method of generating immobilized complements of a plurality of target polynucleotides, the method comprising: a) contacting the solid support of claim 1 with a sample comprising a plurality of target polynucleotides and annealing two or more target polynucleotides to a first plurality of first oligonucleotides; b) extending the first oligonucleotides with a polymerase to generate a plurality of immobilized complements of the plurality of target polynucleotides; c) annealing the immobilized complements of the target polynucleotides to the second plurality of immobilized oligonucleotides; and d) extending the second plurality of immobilized oligonucleotides thereby generating a plurality of immobilized target polynucleotides.

17 . The method of claim 16 , further comprising contacting the immobilized target polynucleotide with a sequencing primer, extending the sequencing primer to incorporate a detectable label that indicates the identity of a nucleotide in the target polynucleotide, detecting the detectable label, and repeating the extending and detecting steps.

18 . A kit comprising a solid support comprising: a first oligonucleotide attached to said solid support comprising a first sequence 251capable of hybridizing to a first endogenous sequence of a target polynucleotide; and

a second oligonucleotide attached to said solid support comprising a second sequence capable of hybridizing to the complement of a second endogenous sequence of said target polynucleotide.

19 . A multiwell container comprising two or more wells, wherein one or more wells comprise a plurality of immobilized oligonucleotides capable of binding target polynucleotides comprising an NRAS gene, or a portion thereof, a MLF2 gene, or a portion thereof, a KRAS gene, or a portion thereof, a TP53 gene, or a portion thereof, a ERBB2 gene, or a portion thereof, a IDH1 gene, or a portion thereof, a PIK3CA gene, or a portion thereof, a KIT gene, or a portion thereof, a PIK3R1 gene, or a portion thereof, a NPM1 gene, or a portion thereof, a EGFR gene, or a portion thereof, a BRAF gene, or a portion thereof, and/or a JAK2 gene, or a portion thereof;

wherein one or more of said immobilized oligonucleotides comprise:

a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 7; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 9; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:11; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:13; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 15; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:17; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:19; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:21; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:23; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:25; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:27; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:29; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 31; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:33; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:35; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:37; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:39; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:41; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:43; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:45; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:47; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:49; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:51; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 53; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:55; at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:57; and/or at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:59.

20 . The multiwell container of claim 19 , wherein said immobilized oligonucleotides comprise sets of oligonucleotides, wherein each set comprises a first sequence and a second sequence selected from:

the first sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO:7; and the second sequence comprises a sequence that is at least 80% identical to a continuous 30 nucleotide sequence within SEQ ID NO: 8;

Assignments (2)

SECURITY INTEREST Recorded Mar 7, 2025

From: SINGULAR GENOMICS SYSTEMS, INC.

To: FIRST-CITIZENS BANK & TRUST COMPANY

Reel/Frame 070440/0465 →

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 20, 2024

From: GLEZER, ELI N.; WITTERS, DAAN; LE, TUNG THANH

To: SINGULAR GENOMICS SYSTEMS, INC.

Reel/Frame 067469/0866 →